Agric. sci. dev., Vol(3), No (5), May, 2014. pp.

194-199

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Agriculture Science Developments

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ISSN:
2306-7527
Copyright 2014. All rights reserved for TI Journals.

Responses of Two Figs (Ficuscarica L.)Cultivars under Salt Stress via

In Vitro Condition
Rohollah AbdoliNejad
Department of Horticulture, College of Agriculture, Shiraz University, Shiraz, Iran.

Akhtar Shekafandeh*
Department of Horticulture, College of Agriculture, Shiraz University, Shiraz, Iran
*Corresponding author: shekafan@shirazu.ac.ir

Keywords
Antioxidant enzymes
Ficus carica
Salt stress
Proline

1.

Abstract
In this research, the effects of different salt concentration (0, 80, 120 and 180 mM NaCl) on physiological
and biochemical changes of two fig cultivars Anjir Sabz and Shah Anjir were evaluated via in vitro
culture. A factorial experiment of 4(levels of salt) 2 (cultivars) in a completely randomized design with 4
replicates and 3 seedlings in each replication was used. The results showed that the concentrations of Na +
and Cl- in leaves and roots of treated seedlings were increased as salt concentration in culture media
increased, but toxicity effect of Cl- was more important in Shah Anjir than Anjir Sabz. The same level of
salinity had less destructive effect on chlorophyll degradation of Shah Anjir. In both cultivars, with
increasing the salt concentration in the culture media, proline content and the activity of antioxidant
enzymes increased, but the amount of total protein decreased. Proline accumulation and the activity of APX
enzyme were significantly higher and the activity of CAT was lower in Shah Anjir than Anjir Sabz.

Introduction

Salinityisan important factor that restricts plant growth and crop production. Thus, the assessment of the existing and/or newly developed
germplasm of plants for their tolerance to salinity is essential. In vitro culture is an ideal system for defining the genetic potential of woody
plants, as it can be carried out under controlled conditions with limited space and time [23, 34]. Such systems can provide appropriate
information about physiological and biochemical responses of plant species, especially if the in vitro induced responses mimic the in vivo plant
responses, exposed to same salinity levels [34]. Salt stress increases the rate of reactive oxygen species (ROS) via enhanced seepage of electron
to oxygen in the chloroplasts, mitochondria and peroxisomes [11]. It is obvious that salinity leads to the closure of stomata in leaves. This
reduces the availability of CO2 in leaves and prohibits the capture of CO2 exposing chloroplasts to energy deficits and oxidative stress. Taking
into account that Cl- is directly involved to the electron transport chain during H 2O oxidation, it is probable that Cl- toxicity interrupts normal
flow of electrons in photosystem II [14] and leads to an increased production of reactive oxygen radicals (ROS) and consequently causes
oxidative stress [30]. Plants evolved protective enzymatic and non-enzymatic mechanisms to scavenge ROS and to alleviate their deleterious
effects. The enzymatic mechanism includes superoxide dismutase (SOD), a metallo-protein that catalyzes the change of superoxide radicals into
hydrogen peroxide. To prevent the accumulation of hydrogen peroxide a compound even more harmful than the superoxide radical the two
enzymatic activities of catalase(CAT)and ascorbate peroxidase(APX) cause dissociation of hydrogen peroxide to O2 and H2 O [16, 21, 22].
Catalase (CAT) is foundprimarilyin the peroxisomes, where the H 2O2 produced during photorespiration and -oxidation of fatty acidsare
metabolized. Peroxidase (POD) belongs to a group of enzymes that reduce H 2O2 to H2O. Ascorbate peroxidases (APX) are antioxidants that
have functions similar to catalase. However, unlike catalase, they aid in the removal of H2O 2 via the utilization of ascorbate as a reductant. APX
have an important role in regulating intracellular levels of H 2O2 in higher plants [28, 32]. Proline is accumulated by a number of different plants
in response to abiotic stresses (water deficit and salinity). There is also strong evidence to suggest that proline metabolism has a significant effect
on cellular redox potential, which may be important for signaling processes associated with salinity tolerance [17]. The increased activities of the
enzymes could reflect a defense response to the cellular damage stimulated by high salt concentrations in the culture media.Ashraf and Harris [2]
mentioned that plants containing high antioxidants show considerable tolerance to oxidative damage caused by ROS. Sorkheh et al.[29]
demonstrated that antioxidant enzyme levels could be used as specific markers for studying salt tolerance and variance in salinity tolerance
among almond species. Fig trees (FicuscaricaL.) a member of the Moraceae family is native to Asia and the eastern Mediterranean region [10].
Iran stands fourth in terms of fig production [13]. Due to uninterrupted years of drought, soil salinity has been intensified and constitutes a major
constraint for Iran fig production industry. As a result, and due to the programs of developing fig orchards, the salinity issue has received notable
attention. Traditionally, fig trees are propagated by stem cutting. Now, orchardists show a tendency for grafted fig trees. As far as we know, fig
rootstocks have not yet been evaluated and characterized for salt tolerance.
The objective of this research was to evaluate the fluctuations in the contents of antioxidant enzymes, proline and total proteins of two fig
cultivars in response to salt stress via in vitro culture for assessment of their salt tolerance.

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Responses of Two Figs (Ficus carica L.) Cultivars under Salt Stress via In Vitro Condition
Agriculture Science Developments Vol(3), No (5), May, 2014.

2.

Methodology

The fruits of two fig cultivars, Anjir Sabz and Shah Anjir were collected from openly pollinated fig trees in the Estahban Fig Research
Station in Fars Province, A total of 1000 seeds of Anjir Sabz and Shah Anjir were sown in metallic containers filled with sandplacing under
mist system. When seedlings reached in a stage of 6 leaves, they were fed with a commercial fertilizer (Kristalon) every two weeks. After 8
month, the same seedlings about 30 cm in length were used in the experiment. Murashige and Skoog [24] medium supplemented with sucrose
(30g L-1) was used as culture medium. The pH of the medium was adjusted to 5.8 before autoclaving (they were autoclaved at 121C for 20
min). The seedlings (30 cm height) were washed thoroughly with tap water, and then they were soaked in 2% solution of a fungicide (Benolit)
for 10 min. After that, they were sterilized using a commercial bleach (Clorax) containing 5.25% NaOCl for 10 min and rinsed 4 times with
sterilized distill water. For stimulation of new shoot growth, the leaf pruned seedlings were transferred in jars of 500 ml that contained 400 ml of
MS medium so that their roots submerged in MS medium in the Jars and their shoot were out of the Jars and they were kept tightly by the help of
the lid, cotton and aluminum foil and media were aerated by help of air pump. The media were refreshed each week. The cultures were
maintained at 251C under a 16-h photoperiod (white light, 51 mol m-2s-1). After 1 month, when seedlings were well established and new
growth started, four different concentrations of NaCl (0, 80, 120 and 160 mM) were added to the medium. After 6 weeks of treatments the data
were recorded.
2.1 Mineral content
Leaf and root Na+, K+ and Cl- content were measured based on Chapman and Pratt methods [7].
2.2 Total chlorophyll
Chlorophyll was extracted from leaf discs in 80% ethanol and the yielded solution was centrifuged at 8000 rpm for 10 min. Chlorophyll content
was colorimetrically determined using the following formula [1]:
Chlorophyll (mg g-1 fresh weight) = [20.2 (A 645) + 8.02 (a 663) x V] / (1000 w)
Where A is absorption value, V is ultimate volume of extract and W is leaf fresh weight.
2.3 Proline and Protein content
Proline was determined by the method of Bates et al. [4]. Leaf tissues were homogenized in 3% sulfo-salicylic acid and after centrifuge at
3000g for 20 min, the supernatant was treated with acetic acid and ninhydrin, and then the absorbance was determined at 520 nm. Proline
(SigmaTM) was used for a standard curve. The protein concentrations of leaf crude extract were determined according to Bradford [5].
2.4 Antioxidant Enzyme activity
Frozen leaf samples (0.5 g) were used for enzyme extraction. Samples were ground in 2 ml of 50 mM phosphate buffer (pH 7.2) using prechilled mortar and pestle. The phosphate buffer contained 1mM EDTA, 1mM PMSF and 1% PVP-40. Then, the extract was centrifuged at 4C
at 17,000g for 10 min. The supernatant was used for measurements of enzyme activity. Superoxide dismutase (SOD EC 1.15.1.1) activity was
measured using Giannopolitis and Reis method [15]. The reaction solution (3 ml) contained 50 M NBT, 1.3 M riboflavin, 13 mM methionine,
75 mM EDTA, 50 mM phosphate buffer (pH 7.8), and 20-50 l of the enzyme extract. The test tubes containing the reaction solution were
irradiated under artificial light (15 fluorescent lamps) at 78 mol m-2s-1 for 15 min. The absorbance of the irradiated solution was read at 560 nm
using a spectrophotometer (Cary 50). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% -nitro blue tetrazolium
chloride (NBT) photo-reduction. Catalase (CAT, EC 1.11.1.6) activity was measured using the method of Chance and Maehly [6]. The assay
solution contained 50 mM potassium phosphate buffer (pH 7.0) and 15 mM H 2O2. The reaction was started by the addition of 100 liters of
enzyme extract to the reaction mixture and the change in absorbance ensued 1 min after the start of the reaction. One unit of activity was
considered as the amount of enzyme that decomposes 1 mM of H2O 2 in one minute. Peroxidase (POD, EC 1.11.1.7) activity was determined by
measuring the oxidation of guaiacol in the presence of H 2O2, and following an increase in absorbance at 470 nm over a 1-min interval. The
enzyme was assayed in a solution containing 50 mM phosphate buffer (pH 7.0), 0.3% H 2O2and 1% guaiacol. The reaction started by the addition
of 20 l enzyme extract at 25C. One enzyme unit was calculated on the basis of the formation of 1mM tetra-guaiacol for 1 min. APX (EC
1.11.1.11) activity was estimated by monitoring the decline in absorbance at 240 nm according to Nakano and Asada [25]. Each 3 ml reaction
medium contained: 50 mM K-phosphate buffer, (pH 7.0), 0.1 mM H 2O2and 20 l enzyme extract. The amount of ascorbate oxidized was
calculated using the extinction coefficient of 2.8 mM-1cm-1and the activity was expressed as mM ascorbate oxidized mg-1 protein min-1.
2.5 Statistical analysis
A factorial experiment 4 2 was used in a complete randomized design with four replicates. The factors were four levels of salinity and two fig
cultivars. Each replicate incorporated three seedlings. The data analysis was performed by SAS 9.1 software. The means were compared using
LSD test at 5% of probability.

3.

Results

Contents of section 3 goes here. It might include some subheadings. Format of subheadings The results showed that with increasing salinity in
external media the rate of Na+ and Cl-accumulation in leaves and roots of both cultivars increased; however leaf sodium content in 80 and 120
mMNaCl in Shah Anjir was significantly higher than AnjirSabz. A high interaction was observed between salt and cultivars on leaf Clcontent, in 80 mMNaCl Shah Anjir showed more Cl- content than AnjirSabz, but in 160mM NaCl, the rate of accumulation of Cl- in
AnjirSabz was more than Shah Anjir(Table 1).

Rohollah Abdolinejad, Akhtar Shekafandeh *

196

Agriculture Science Developments Vol(3), No (5), May, 2014.

Table1. Interaction of salt and cultivar on Na+ and Cl - (% fresh weight) contents of the leaves and the roots

NaCl
mM
0 (Control)
80
120
160
significance
NaCl
Cultivar
NaCl Cultivar

Leaf Na+
Anjir
Shah
Sabz
Anjir
0.01f
0.02f
0.25e
0.39d
0.66c
0.77b
1.06a
0.99a

Root Na+
Anjir
Shah
Sabz
Anjir
0.03d
0.04d
0.39c
0.10d
0.83b
0.70b
1.21a
1.16a

Leaf ClAnjir
Shah
Sabz
Anjir
0.79f
0.73f
1.94e
2.49d
3.08c
3.21c
4.51a
4.28b

**
ns
ns

**
**
ns

**
*
**

Root ClAnjirS Shah

abz
Anjir
0.57e
0.55e
1.14c
0.99d
1.89b
1.74b
2.25a
2.30a
**
ns
ns

In each subject, means with the same letters are not significantly different atP 0.05 using LSD test. * P 0.05. ** P 0.01

The leaf and root K+ contents decreased in both cultivars with increasing NaCl in the media, but in each level of salinity there was not significant
between two cultivars except in control in which the root k+ content of Shah Anjir was more than Anjir Sabz. In both cultivars, the leaf total
chlorophyll was significantly enhanced in low level of salinity (80 mM NaCl), but with increasing 120 mM NaCl in culture media total
chlorophyll decreased, but this reduction was more pronounced in Anjir Sabz (Table 2).
Table 2. Interaction of salt and cultivar on leaf and root K+ content (% fresh weight) and on leaf chlorophyll (mg g-1
F.W) content

NaCl
mM
0 (Control)
80
120
160
significance
NaCl
Cultivar
NaCl Cultivar

Leaf K+
AnjirSabz Shah
Anjir
3.62a
3.74a
3.39b
3.37b
2.75c
2.63c
2.21d
2.11d

Root K+
AnjirSabz Shah
Anjir
2.46b
2.62a
2.51b
2.39bc
2.21d
2.32cd
1.97e
2.06e

**
ns
*

**
ns
*

Chlorophyll
AnjirSabz Shah
Anjir
16.75b
15.27bc
20.75a
19.50a
14.10cd
17.01b
11.58e
12.44de
**
ns
*

In each subject, means with the same letters are not significantly different atP 0.05 using LSD test. * P 0.05. ** P
0.01

In relation to leaf proline, with increasing salt concentration in the media, the synthesis and accumulation of proline increased markedly (Table
3). The highest proline accumulation was observed in 160 mM NaCl, whereas the lowest concentration occurred in the control. Regardless of
salinity, Shah Anjir accumulated significantly more proline (93.18 mol g-1 F.W) than Anjir Sabz (82.81 mol g-1 F.W). In both cultivars,
parallel to the increase of salt concentration, the amount of protein decreased (Table 4).
Table 3. Effect of salt stress on leaf proline content (mol g-1 F.W) of two fig cultivars.

Cultivar
AnjirSabz
Shah Anjir
Mean
Significance
NaCl
Cultivar
NaClCultivar

0
41.15 d
39.77 d
40.46D

80
58.35 c
64.42 cd
61.28 C

NaCl (mM)
120
160
58.35 c
132.07 a
64.42 cd
141.47 a
112.38 B
136.77 A
proline (mol g-1 F.W)
**
*
**

Mean
82.81 B
93.18 A

Means that have the same letters (small for interactions and capital for main effects) are not significantly different at P 0.05 using LSD
test. * P 0.05. ** P 0.01

Table 4. Effect of salt stress on leaf total protein content (mol g-1 F.W) of two fig cultivars.

Cultivar
AnjirSabz
Shah Anjir
Mean
Significance
NaCl
Cultivar
NaClCultivar

0
114.25 b
129.35 a
121.80 A

80
103.65 bcd
108.95 bc
106.30 B

NaCl (mM)
120
160
96.05 d
79.25 e
100.55 cd
93.40 d
98.30 C
86.22 D
proline (mol g-1 F.W)
**
*
**

Mean
98.30 B
108.06 A

Means that have the same letters (small for interactions and capital for main effects) are not significantly different at P 0.05 using LSD test.
* P 0.05. ** P 0.01

In all levels of salinity Shah Anjir showed higher amount of protein in comparison with Anjir Sabz. In general, when the salt concentration
in the culture media increased, a significant rise in SOD activity was achieved (Table 5). In higher levels of salinity (120 and 180 mM NaCl) the

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Responses of Two Figs (Ficus carica L.) Cultivars under Salt Stress via In Vitro Condition
Agriculture Science Developments Vol(3), No (5), May, 2014.

rate of SOD activity in Shah Anjir was relatively greater than Anjir Sabz, although there were not significant differences between them.
There was a high interaction between salinity and cultivar on CAT activity. In low level of NaCl (0 and 80 mM) Anjir Sabz showed
significantly higher activity of CAT, however in higher levels, Shah Anjir increased its CAT activity, so that there was not significant
differences between two cultivars (Table 6). The highest POD enzyme activity (69.94) was observed in 120 mM NaCl in Anjir Sabz which
was significantly higher than Shah Anjir in the same treatment, but, in 80 mM NaCl Shah Anjir showed greater POD activity than Anjir
Sabz(Table 7). Salinity enhanced Apex activity in leaves of both cultivars (Table 8). The maximum APXactivity was detected in 160 mM NaCl
which was 6.8 and 3.8 times higher compared to their control respectively for Shah Anjir and Anjir Sabz.
Table 5. Effect of salt stress on SOD activity (U g-1 F.W) of two fig cultivars.

NaCl (mM)
Cultivar
0
80
120
160
Mean
Anjir Sabz
128.50 d
130.50 d
263.50 c
403.00 ab
231.38 A
Shah Anjir
87.00 d
122.00 d
319.50 bc
473.50 a
250.50 A
Mean
107.75 C
126.25 C
291.50 B
403.00 ab
Significance
SOD (U g-1 F.W)
NaCl
**
Cultivar
ns
NaClCultivar
*
Means that have the same letters (small for interactions and capital for main effects) are not significantly different at P 0.05
using LSD test. * P 0.05. ** P 0.01
Table 6. Effect of salt stress on CAT activity (U g-1 F.W) of two fig cultivars

Cultivar
Anjir Sabz
Shah Anjir
Mean
Significance
NaCl
Cultivar
NaClCultivar

0
31.57 cd
16.63 e
24.10 C

80
36.43 bc
22.84 de
29.64 C

NaCl (mM)
120
46.19 b
41.00 bc
43.59 B
CAT (Ug-1 F.W)
**
*
**

160
59.71 a
59.99 a
59.85A

Mean
43.47 A
35.12 B

Means that have the same letters (small for interactions and capital for main effects) are not significantly different at P 0.05 using LSD test.
* P 0.05. ** P 0.01

Table 7. Effect of salt stress on POD activity (U g-1 F.W) of two fig cultivars

Cultivar
AnjirSabz
Shah Anjir
Mean
Significance
NaCl
Cultivar
NaClCultivar

NaCl (mM)
0
12.04 e
15.79 e
13.91 C

80
28.12 d
37.95 c
33.03 B

120
69.94 a
58.76 b
64.35 A
POD (U g-1 F.W)
**
*
**

160
41.18 c
36.07 cd
38.62 B

Mean
37.82 A
37.14 A

Means that have the same letters (small for interactions and capital for main effects) are not significantly different at P 0.05 using LSD test.
* P 0.05. ** P 0.01

4.

Discussion

Three days after the plants were treated with different levels of salt, marginal chlorosis was noted on the leaves in 120 and 160 mM NaCl which
later resulted in necrosis. These symptoms were less prominent in Shah Anjir than AnjirSabz. In both cultivars, any chlorosis symptoms were
not observed in 80 mM NaCl.
It is well known that the rate of photosynthesis reduces with stress, accompanied by degradation of chlorophyll and chlorophyll-protein complex
[9]. In our study in both cultivars the total chlorophyll concentration significantly increased in 80 mM NaCl compared with control and then
diminished in higher levels of salt, but this reduction in Shah Anjir was lower than Anjir Sabz. This shows that the same level of salt had less
destructive effect on chlorophyll degradation in Shah Anjir. On the other hand, leaf Na+ content in 80 and 120 mM NaCl in Shah Anjir was
more than Anjir Sabz. It seems that Shah Anjir used Na+ for osmotic regulation [26]. Leaves necrosis was more intense in Anjir Sabz than
Shah Anjir (especially in 160 mM NaCl) which may be due to toxicity effect of Cl- (Table 1). Totally, Positive correlations were found
between leaf proline levels and salt ions (Na+: r = 0.918 p<0.001 and Cl-: r = 0.881 p<0.001). It is well documented that following salt, drought
and metal stress there is a dramatic accumulation of proline may be due to increased synthesis or decreased degradation. Free proline has been
proposed to act as an osmoprotectant, a protein stabilizer, a metal chelator, an inhibitor of lipid peroxidation and OH_ scavenger [3, 31, 33].
Increased accumulation of proline has been correlated with improved tolerance to various abiotic stresses especially salt and drought. Enhanced
synthesis of proline under drought or salt stress has been implicated as a mechanism to alleviate cytoplasmic acidosis and maintain
NADP:NADPH at values compatible with metabolism [18].

Rohollah Abdolinejad, Akhtar Shekafandeh *

198

Agriculture Science Developments Vol(3), No (5), May, 2014.

It has been shown that as the salt concentration increased, the total amount of soluble protein in the leaves of two tobacco cultivars declined
under both in vitro and in vivo conditions [19]. The evaluation of olive cultivars under salt-stress showed that there was a correlation between
the fluctuations in protein and proline content of the olive leaves. Our results confirmed the above findings, and it seems that the reduction in
protein content under stressful conditions is related to its lessened synthesis, the increase in the amount of enzymes responsible for protein
degradation and the buildup of free amino acid, including proline [2].
In both fig cultivars, the activity of SOD enzyme increased with increasing external medium salt. It appears that a salt concentration of 160 mM
is not a limiting factor for the formation and activity of the SOD enzyme. The raise in activity of the SOD enzyme has been reported by elik
and Atak [19] in two Turkish tobacco cultivars and by Ertruk et al. [12] in the cherry rootstock 'Gisela 5', under both in in vitro and in vivo
conditions.
In high level of salinity (160 mM NaCl) CAT activity was about 1.89 and 3.6 times higher in Anjir Sabz and Shah Anjir respectively in
comparison with their controls. Catalase is an important and influential anti-oxidant in the defense system of most plants that acts against abiotic
stresses. This enzyme can directly degrade hydrogen peroxide to oxygen and water molecules, thereby eliminating the toxicity of hydrogen
peroxide [27]. The degree of CAT enzyme activity also increased in the cherry rootstock Gisela 5 as the salt concentration increased in the
culture media [12].
The peroxidase enzyme (POD), with various forms and structures, is responsible for eliminating hydrogen peroxide from biological systems
[20]. In the same manner, the activity of POD in Prunus cerasus leaves increased when NaCl and CaCl2 were added to the MS medium [8].
Ascorbic peroxidase is also another important antioxidant in plants that protects higher plant cells from stresses through its removal of free
oxygen radicals [28]. Ascorbic peroxidase (APX) has five different iso-forms which can be found in cell membranes, the cytoplasm and
thylakoid. The increase in the activity of the APX enzyme in the leaves of two Turkish tobacco cultivars has been reported by elik and Atak
[19] with increasing salt concentrations in media under in vitro conditions. We obtained similar results with two fig cultivars.

5.

Conclusion

In both genotypes, the concentrations of Na+ and Cl- in leaves and roots of treated seedlings were increased as salt concentration in culture media increased. Shah
Anjir resisted more against toxicity effect of Cl- and showed less chlorophyll degradation than Anjir Sabz. In both cultivars, with increasing the salt
concentration in the culture media, proline content and the activity of antioxidant enzymes increased. Proline accumulation and the activity of APX enzyme were
significantly higher and the activity of CAT was lower in Shah Anjir than Anjir Sabz.

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